Synthetic design of S. cerevisiae ChrVII: 1 048 595 – 1 078 595 Frederico Magalhães, Kristoffer Krogerus

Region The right arm of chromosome VII (1 048 595 – 1 078 595) ORFs in this region: SCW4 ZUO1 MAL1x locus PXR1 BIO2 MAL11 MAL12 YOR1 IMA1 MAL13 BGL2 YGR290W YGR283C YGR291C ERV29

MAL1x locus One of many loci that enables maltose utilization Three gene complex MAL11: Maltose permease MAL12: Maltase MAL13: MAL activator Essential phenotype for many industrial applications: Beer fermentation Bioethanol fermentation Baking industry Maltose Maltose Glucose Maltase

Region Essential ORFs in this region: SCW4 BIO2 PXR1 IMA1 YOR1 MAL1x locus BGL2 MAL11 YGR283C MAL12 MAL13 ERV29 (YGR290W) ZUO1 YGR291C To maintain: -PXR1: Essential protein involved in rRNA and snoRNA maturation -BGL2: Endo-beta-1,3-glucanase; major protein of the cell wall, involved in cell wall maintenance -ERV29: Protein localized to COPII-coated vesicles; involved in vesicle formation and incorporation of specific secretory cargo -ZUO1: Ribosome-associated chaperone; zuotin functions in ribosome biogenesis and as a chaperone for nascent polypeptide chains in partnership with Ssz1p and SSb1/2 -BIO2:Biotin synthase; catalyzes the conversion of dethiobiotin to biotin, which is the last step of the biotin biosynthesis pathway -IMA1:Major isomaltase (alpha-1,6-glucosidase/alpha-methylglucosidase); required for isomaltose utilization -MAL Effect of deletion must be clarified: -SCW4:Cell wall protein with similarity to glucanases; scw4 scw10 double mutants exhibit defects in mating -YGR283C:Putative methyltransferase; may interact with ribosomes Can be deleted: -YOR1: Plasma membrane ATP-binding cassette (ABC) transporter -YGR290W*: Dubious open reading frame; unlikely to encode a functional protein. *partially overlaps with MAL11 -YGR291C: Dubious open reading frame; unlikely to encode a functional protein Removing the ORFs (maltose-rich media) No effect Slightly reduced growth and viability Reduced growth and viability

Alterations Removal of non-essential genes Critera: no effect on growth or viability in industrial settings ORFs did not contain any introns Replace stop codons (TAG > TAA) Add PCRtags to ORFs Add loxP sites to 3’ UTR of SCW4 and YGR283C, and in place of YOR1 and YGR291C

Workflow Retrieve sequences from SGD (www.yeastgenome.org) Replace stop codon in all ORFs (TAG > TAA) Insert PCRTags using ’Codon Juggle’ and ’most different’-algorithm from GeneDesign tool (http://genedesign.jbei.org/) Design primers and check specificity (e.g. Primer3; http://simgene.com/Primer3) Insert loxP sites Design ~10 kB megachunks Linkers on both side (allow integration by recombination) Potential antibiotic resistance marker for selection

Example of PCRTag addition Switch codons in the ORF, such that the amino acid sequence stays the same, but the DNA sequence is different (at least two locations of ~ 20 bp) PCR primer pairs can then be designed for these locations such that the synthetic and wild type sequences can be selectively differentiated. Target areas with serine, arginine, and leucine E.g. MAL13: Wild-type: Altered:

Example of loxP site addition Add in the 34 bp sequence for the wild-type loxP site in the 3’ untranslated region (i.e. 3 bp following the stop codon) Recognition region Spacer region ATAACTTCGTATA ATGTATGC TATACGAAGTTAT ORF … NNNTAANNNNN … … NNNTAANNNATAACTTCGTATAATGTATGCTATACGAAGTTATNN …

Construction – 3 megachunks loxP site

Construction YPD + hygromycin hphMX Hygromycin resistance Linker hphMX

Construction YPD + YPD + Nourseothricin resistance Linker hygromycin hphMX YPD + hygromycin YPD + nourseothricin natMX4 Nourseothricin resistance Linker natMX4

Construction natMX4 YPD + nourseothricin YPD Linker

Final synthetic region Size: 25 503 bp 4 loxP sites 2 ORFs removed PCRtags to distinguish wild-type and synthetic genes ORFs (1-10) WT strain loxP site SynVII strain (adapted from Dymond et al. 2011, Nature)

Suggestions for future work Additional deletions: The whole MAL locus could be eliminated if a maltase that is secreted was introduced: Bacillus subtilis Bacillus brevis Maltose Glucose Maltase